Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1998-12-22
2002-11-19
Graser, Jennifer E. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S265100, C424S185100, C424S191100, C530S300000, C530S350000
Reexamination Certificate
active
06482415
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to detecting exposure to microorganisms by the use of serodiagnostic assays, and more specifically to detecting exposure to
Orientia tsutsugamushi.
2. Description of Prior Art
Scrub typhus or tsutsugamushi disease is an acute, febrile disease caused by infection with
Orientia
(formerly Rickettsia)
tsutsugamushi.
It accounts for up to 23% of all febrile episodes in endemic areas of the Asia-Pacific region (5). The incidence of disease has increased in some countries during the past several years (6).
O. tsutsugamushi
is a gram negative bacterium, but in contrast to other gram negative bacteria,
O. tsutsugamushi
has neither lipopoly-saccharide nor a peptidoglycan layer (1) and the ultrastructure of its cell wall differs significantly from those of its closest relatives, the typhus and spotted fever group species in the genus Rickettsia (33). The major surface protein antigen of
O. tsutsugamushi
is the variable 56 kDa protein which accounts for 10-15% of its total protein (16, 28). Most type-specific monoclonal antibodies to Orientia react with homologues of the 56 kDa protein (16, 24, 42). Sera from most patients with scrub typhus recognize this protein, suggesting that it is a good candidate for use as a diagnostic antigen (28).
Diagnosis of scrub typhus is generally based on the clinical presentation and the history of a patient. However, differentiating scrub typhus from other acute tropical febrile illnesses such as leptospirosis, murine typhus, malaria, dengue fever, and viral hemorrhagic fevers can be difficult because of the similarities in signs and symptoms. Highly sensitive polymerase chain reaction (PCR) methods have made it possible to detect
O. tsutsugamushi
at the onset of illness when antibody titers are not high enough to be detected (14, 19, 36). PCR amplification of the 56 kDa protein gene has been demonstrated to be a reliable diagnostic method for scrub typhus (14, 18). Furthermore, different genotypes associated with different Orientia serotypes could be identified by analysis of variable regions of this gene without isolation of the organism (14, 17, 18, 25, 39). However, gene amplification requires sophisticated instrumentation and reagents generally not available in most rural medical facilities. Current serodiagnostic assays such as the indirect immunoperoxidase (IIP) test and the indirect immunofluorescent antibody (IFA) or microimmunofluorescent antibody (MIF) tests require the propagation of rickettsiae in infected yolk sacs of embryonated chicken eggs or antibiotic free cell cultures (4, 20, 30, 43).
At the present time the only commercially available dot-blot immunologic assay kits (Dip-S-Ticks) requires tissue culture grown, Renografin density gradient purified, whole cell antigen (41). Only a few specialized laboratories have the ability to culture and purify
O. tsutsugamushi
since this requires biosafety level 3 (BL3) facilities and practices. The availability of recombinant rickettsial protein antigens which can be produced and purified in large amounts and have similar sensitivity and specificity to rickettsia-derived antigens would greatly reduce the cost, transport, and reproducibility problems presently associated with diagnostic tests which require the growth and purification of rickettsiae.
Recently, a recombinant 56 kDa protein from Boryong strain fused with maltose binding protein was shown to be suitable for diagnosis of scrub typhus in a enzyme-linked immunosorbent assay (ELISA) and passive hemagglutination test (21, 22). Although this protein overcomes some of the above-described disadvantages, it still has certain inherent disadvantages as an assay reagent because it is a fusion protein.
SUMMARY OF THE INVENTION
Accordingly, an object of this invention is a recombinant construct and expressed polypeptide possessing immunogenic regions.
Another object of the invention is a recombinant polypeptide encoding a portion of the 56 kDa protein of
O. tsutsugamushi
encoded by amino acids 80 to 456.
A still further object of the invention is a recombinant truncated 56 kDa polypeptide which is re-folded to give a soluble moiety.
An additional object of this invention is the use of the recombinant polypeptide in antibody based assays for improved methods for the detection of
O. tsustugamushi
exposure, in research and in clinical samples.
Yet another object of the invention the expression of the truncated r56 in different host backgrounds of bacterial strains for use in different vaccine formulations against scrub typhus infection.
REFERENCES:
Stover et al. Infect. Immun. 1990. 58(7): 2076-2084.*
Kim et al. J. Clin. Microbiol. 1993, 31(3): 598-605.*
Ohashi et al. Infect. Immun. 1989. 57(5): 1427-1431.*
Ohashi et al. Gene. 1990. 91: 119-122.*
Ohashi et al. J. Bio. chem. 1992. 267(18): 12728-12735.
Ching Wei-Mei
Dasch Gregory A.
Kelly Daryl J.
Graser Jennifer E.
Hemby, Jr. Joseph K.
Spevack A. David
The United States of America as represented by the Secretary of
LandOfFree
Expression and refolding of truncated recombinant major... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Expression and refolding of truncated recombinant major..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Expression and refolding of truncated recombinant major... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2919810